European Journal of Cardiovascular Medicine

The emergence of antimicrobial resistance in Gram-negative pathogens poses a serious global threat to public health, particularly in the treatment of bloodstream infections (BSIs). Among these pathogens, members of the family Enterobacteriaceae are of particular concern due to their ability to acquire and disseminate resistance determinants rapidly. Colistin, a last-resort polymyxin antibiotic, has regained clinical significance for the treatment of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Enterobacteriaceae [1]. However, the increasing reports of colistin resistance have greatly undermined its clinical utility [2].

Resistance to colistin was historically attributed to chromosomal mutations affecting the pmrAB, mgrB, and phoPQ regulatory systems [3]. However, the identification of plasmid-mediated colistin resistance genes—particularly mcr-1 and its variants—has marked a paradigm shift in our understanding of resistance transmission [4]. First reported in Escherichia coli from animal and human samples in China in 2015, the mcr-1 gene has since been detected globally across various species of Enterobacteriaceae [5].

The mcr-2 gene, although less prevalent, similarly encodes a phosphoethanolamine transferase that modifies the bacterial lipid A structure, reducing colistin binding and efficacy [6].

The plasmid-borne nature of mcr genes facilitates horizontal gene transfer between bacterial strains and species, increasing the potential for widespread dissemination in both community and hospital settings [7]. Bloodstream infections caused by mcr-positive organisms are particularly worrisome due to their high morbidity and mortality rates, limited treatment options, and diagnostic challenges. Accurate detection and molecular characterization of these resistance determinants are critical to inform empirical therapy, guide infection control strategies, and support antimicrobial stewardship initiatives [8].

In India, where the burden of antimicrobial resistance is among the highest in the world, the surveillance of mcr-mediated colistin resistance remains underdeveloped, especially in bloodstream isolates. There is a pressing need for regional data to understand the epidemiology, molecular characteristics, and clinical implications of mcr-positive Enterobacteriaceae in tertiary care settings.

This study aims to detect the presence of mcr-1 and mcr-2 genes in Enterobacteriaceae isolated from bloodstream infections at IMS & SUM Hospital, Bhubaneshwar, Odisha, and to characterize the phenotypic and genotypic resistance patterns associated with these isolates.

Aims and Objectives

Aim:

To detect and characterize colistin resistance mediated by mcr-1 and mcr-2 genes in Enterobacteriaceae isolated from bloodstream infections.

Objectives:

• To determine the prevalence of phenotypic and genotypic colistin resistance in bloodstream isolates of Enterobacteriaceae using standardized antimicrobial susceptibility testing and PCR-based detection of mcr-1 and mcr-2 genes.
• To correlate the presence of mcr genes with species distribution, antimicrobial resistance profiles, and clinical parameters of the affected patients.

Study Design and Setting:

This was a prospective cross-sectional study conducted over a 12-month period (June 2022 to May 2023) in the Department of Microbiology, IMS & SUM Hospital, Bhubaneshwar, Odisha—a tertiary care teaching hospital in eastern India.

Sample Size and Inclusion Criteria:

A total of 110 non-duplicate Enterobacteriaceae isolates were recovered from blood cultures of hospitalized patients with confirmed bloodstream infections. Only clinically significant isolates with confirmed growth in automated blood culture systems (e.g., BACTEC or BacT/ALERT) were included. Recurrent isolates from the same patient or polymicrobial cultures were excluded.

Phenotypic Antimicrobial Susceptibility Testing:

Isolates were identified to the species level using standard biochemical methods and/or automated identification systems (e.g., VITEK 2 or MALDI-TOF). Antimicrobial susceptibility testing, including colistin MIC determination, was performed using the broth microdilution method following CLSI/EUCAST guidelines. Results were interpreted according to the latest EUCAST breakpoint recommendations for colistin.

Detection of mcr-1 and mcr-2 Genes:

Genomic DNA was extracted from colistin-resistant isolates using a commercial bacterial DNA extraction kit. PCR amplification was performed using specific primers targeting the mcr-1 and mcr-2 genes. Amplification products were visualized via agarose gel electrophoresis, and selected positive amplicons were confirmed by Sanger sequencing where feasible.

Data Analysis:

Clinical and microbiological data were compiled in Microsoft Excel and analyzed using SPSS Version 26.0 (IBM Corp., Armonk, NY, USA). Descriptive statistics were used to summarize resistance patterns and mcr gene prevalence. Associations between genotypic and phenotypic resistance were assessed using the Chi-square or Fisher’s exact test, and p < 0.05 was considered statistically significant.

Phenotypic Resistance to Colistin

Of the 110 Enterobacteriaceae isolates tested, 24 (21.8%) exhibited phenotypic resistance to colistin, as determined by broth microdilution with an MIC >2 µg/mL. Escherichia coli accounted for the largest number of resistant isolates (n = 9), followed by Klebsiella pneumoniae (n = 7), Enterobacter spp. (n = 3), Citrobacter spp. (n = 3), and Serratia spp. (n = 2). The overall susceptibility rate to colistin was 78.2%. Species-wise distribution of colistin resistance is summarized in Table 2.

Detection of mcr-1 and mcr-2 Genes

PCR analysis for colistin resistance determinants revealed that 17 out of 110 Enterobacteriaceae isolates (15.5%) harbored either the mcr-1 or mcr-2 gene. Specifically, mcr-1 was detected in 14 isolates (12.7%) and mcr-2 in 3 isolates (2.7%). Escherichia coli was the most frequently identified mcr-positive species (n = 10), followed by Enterobacter spp. (n = 5), Klebsiella pneumoniae (n = 5), Serratia spp. (n = 2), and Citrobacter spp. (n = 1). The distribution of mcr-positive isolates by species is presented in Table 3. All mcr-positive isolates demonstrated phenotypic resistance to colistin, showing strong concordance between genotypic and phenotypic findings.

Correlation Analysis Between mcr Genes and Colistin MIC

To assess the relationship between colistin MIC values and the presence of mcr genes, a scatterplot analysis was performed. All isolates harboring mcr-1 or mcr-2 demonstrated MIC values exceeding the resistance breakpoint of >2 µg/mL, with values ranging from 4 to 32 µg/mL. In contrast, mcr-negative isolates exhibited lower MIC values, generally within the susceptible range. This analysis indicates a strong phenotypic-genotypic concordance, with significantly elevated MIC values among mcr-positive strains, supporting the reliability of molecular detection in predicting high-level colistin resistance. The MIC distribution by mcr gene status is illustrated in Figure 1.

Figure 1. Colistin MIC distribution by mcr-gene status among bloodstream Enterobacteriaceae isolates (n = 110).
Each point represents the minimum inhibitory concentration (MIC) of colistin for an individual isolate, plotted on a logarithmic scale. Isolates harbouring either mcr-1 or mcr-2 genes (orange, “mcr-positive”) cluster above the EUCAST resistance breakpoint of 2 µg/mL (dashed line), whereas mcr-negative isolates (yellow) fall predominantly within the susceptible range. The clear separation underscores the strong concordance between genotypic detection of mcr genes and phenotypic high-level resistance to colistin.

Table 5: Correlation Between mcr Gene Detection and Phenotypic Colistin Resistance

This cross-sectional study sheds light on the growing threat of colistin resistance among bloodstream Enterobacteriaceae isolates, focusing particularly on the detection of plasmid-mediated resistance genes mcr-1 and mcr-2. Our findings reveal that nearly one in five isolates exhibited phenotypic resistance to colistin, and that 15.5%

harboured either mcr-1 or mcr-2. These figures are particularly concerning given colistin’s status as a last-line antibiotic for treating multidrug-resistant Gram-negative infections [9].

The strong concordance observed between genotypic and

phenotypic resistance—100% of mcr-positive isolates had MICs >2 µg/mL—underscores the clinical relevance of these resistance markers. This is consistent with previously reported studies, where mcr-positive isolates typically demonstrate MICs in the range of 4–32 µg/mL [10]. For instance, Uddin et al. reported widespread mcr-1 positivity in E. coli isolates from poultry in Bangladesh, with sequencing confirming their phenotypic resistance to colistin [11].

In line with our study’s findings, Zhang et al. identified mcr-1 in a significant proportion of E. coli, Klebsiella pneumoniae, and Salmonella species from animal specimens, reinforcing the zoonotic and environmental transmission potential of mcr-mediated resistance [14]. Similarly, Karki et al. documented the presence of mcr-1 among colistin-resistant E. coli and K. pneumoniae in Nepalese clinical settings, reporting a concerning overlap with other resistance mechanisms such as carbapenemases and ESBLs [13].

The predominance of mcr-positive strains among E. coli and K. pneumoniae in our bloodstream isolates mirrors international data [12]. Saavedra et al., in a large retrospective study in Colombia, confirmed that mcr-1 is commonly plasmid-mediated and transferable via conjugation, underscoring the threat of horizontal gene spread in clinical environments [12].

Our results also support the utility of multiplex PCR-based screening, which offers high specificity, faster turnaround time, and reduced dependency on whole genome sequencing for surveillance purposes. Rebelo et al. demonstrated the reliability of multiplex PCR for detecting multiple mcr variants with full concordance to genome sequencing data [10]. Likewise, Osei-Sekyere emphasized the value of combining rapid phenotypic methods such as RPNP with multiplex PCR to improve detection in resource-limited laboratories [11].

One of the strengths of this study lies in its dual phenotypic-genotypic design, supported by scatterplot and cross-tabulation analysis. These allowed us to confirm near-perfect concordance between elevated colistin MICs and mcr gene presence. Such concordance reinforces the role of mcr detection as a reliable diagnostic marker for resistance and may warrant inclusion in future empirical therapy decision-making algorithms

Limitations

Being single-centre and limited to bloodstream isolates, it may not reflect broader regional or environmental patterns. Furthermore, although molecular assays confirmed mcr-1 and mcr-2, we did not assess for newer variants (mcr-3 through mcr-10) or perform plasmid sequencing. Future multicentre studies incorporating whole genome analysis could better elucidate resistance dynamics and transmission pathways.

The detection of mcr-1 and mcr-2 among bloodstream Enterobacteriaceae isolates with concurrent high-level colistin resistance highlights the urgent need for molecular surveillance, antibiotic stewardship, and infection control reinforcement. Timely detection of mcr-positive organisms can play a pivotal role in mitigating the impact of antimicrobial resistance in critical care settings

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